The biological importance of electron-transfer proteins cannot be overstated since life could not exist in the absence of an efficient means of transporting electrons. The objective of this research is to understand on a structural level the mechanisms and control of biological electron transfer in three specific protein systems: the cytochromes C2 which contain heme groups, the high-potential iron-sulfur proteins (HiPIPs) which contain [4Fe-4S] clusters, and the plant-type ferredoxins which contain [2Fe-2S] metal centers. All of these proteins are involved in electron transport and similar mechanisms are thought to modulate the redox properties and electron transfer rates of their respective prosthetic groups. The specific aim of this research is to use a combination of site-directed mutagenesis experiments, X-ray crystallography, and kinetic measurements to test the current explanations for the modulation of the redox properties of the hemes and the iron-sulfur clusters. During the last granting period, the molecular structures of the cytochrome C2 from Rhodobacter capsulatus, the HiPIPs from Ectothiorhodospira halophila and Rhodocyclus tenuis, and the [2Fe-2S] ferredoxins from Anabaena were determined by X-ray crystallographic techniques. By using the technique of site-directed mutagenesis and in consideration of the three-dimensional structures of these molecules, various mutants for each of these classes of proteins will be constructed, their X-ray structures will be solved and their kinetic properties and redox potentials determined. In addition to these mutant proteins, the three-dimensional structures of several other c-type cytochromes and HiPIPs (for which crystals have already been grown) with different biological properties will be determined during the next granting period. These protein systems were chosen because they have been well characterized using both biochemical and biophysical techniques and because the amount of protein necessary for these studies is readily isolated from the organisms. Taken together, these investigations will provide a model for understanding on a quantitative and structural level the modulation of the biological properties for these specific proteins. Furthermore, the knowledge gained from these simpler systems should be directly applicable toward understanding the properties of electron-transfer proteins in higher organisms.